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`and cells were selected for gpt expression. Clones were
`screened by assaying human antibody production in the culture
`supernatant by ELISA, and antibody was purified from the best(cid:173)
`producing clones. Antibody was purified by passing tissue
`culture supernatant over a column of staphylococcal protein A~
`Sepharose CL-4B (Pharmacia). The bound antibodies were e~uted
`with 0.2 M Glycine-HCl, pH 3.0 and neutralized with 1 M Tris
`pH a.o. The buffer was exchanged into PBS by passing over a
`PD10 column (Pharmacia:).
`The binding of the humanized antibodies to cell
`types expressing the corresponding antigens was tested:
`HSV-infected cells for Fd79 and Fdl38-BO, 0937 cells for M195,
`YTJB cells for mik-Pl and CMV-infected cells for CMVS. By
`fluorocytometry, the humanized antibodies bind approximately
`as well as the original mouse antibodies and the corresponding
`chimeric antibodies. Moreover, the humanized antibodies
`compete approximately as well as the corresponding mouse
`antibodies against the radiolabeled mouse antibodies for
`binding to the cells, so the humanized antibodies have
`approximately the same binding affinity as the mouse
`antibodies, typically within about 2-fold or better,~'
`e.g., _'],'able 2.
`
`Binding affinities of murine and humanized antibodies.
`
`TABLE 2
`
`Fd79 (anti-gB)
`
`Fdl38-80 (anti-gD)
`
`Mouse
`
`(M-1)
`
`K
`a
`1.1 X 108
`
`5.2 X 107
`
`Humanized
`
`(M-1)
`
`K
`a
`5.3 X 107
`
`4.8 X 10 7
`
`From the foregoing, it will be appreciated that the
`humanized immunoglobulins of the present invention offer
`numerous advantages over other antibodies.
`In comparison to
`other monoclonal antibodies, the present humanized
`immunoglobulin can be more economically produced and contain
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`substantially less foreign amino acid sequences. This reduced
`likelihood of antigenicity after injection into a human
`patient represents a significant therapeutic improvement.
`A detailed description of each humanized
`immunoglobulin follows.
`
`Example I
`Humanized Immunoqlobulins to p75
`In mammals, the immune response is mediated by two
`types of cells that interact specifically with foreign
`material, i.e., antigens. One of these cell types, B-cells,
`are responsible for the production of antibodies. The second·
`cell class, T-cells, include a wide variety of cellular
`subsets controlling the in vivo function of both B-cells arid a
`wide variety of -other hematopoietic cells, including T-cell~-.
`(See, generally,_ Paul, W.E., ed., Fundamental Immunology, 2nd
`ed., Raven Press, New York .(1989).)
`One way in which T-cells exert this control is
`through the production of a lymphokine known as interleukin-2
`(IL-2) , originally named T-cell growth factor. . IL-2 1 s prime · ..
`function appears to be the stimulation and maintenance of T(cid:173)
`cells.
`Indeed, some immunologists believe that IL-2 may be.at
`the center of the entire immune response(~, Farrar, J., e~
`al., Immunol. Rev. 63, 129-166 (1982)).
`To exert its biological effects, IL-2 interacts ·with'. ·
`a specific high-affinity membrane receptor (Greene, w., et
`al., Progress in Hematology XIV, E. Brown, Ed., Grune and
`Statton, New York (1986}, at pgs. 28.3 ff and Waldmann, &:m.:.
`Rev. Biochem. 58, 875 (1989)). The human IL-2 receptor is·,a
`complex multichain glycoprotein, with one chain, known as the
`Tac peptide or alpha chain, being about 55kD in size (see,
`Leonard~ W., et al._, J. Biol. Chem. 260, 1872 (1985)). The
`second chain is known as the p75 or beta chain (Tsudo et a~.,
`Proc. Nat. Acad. sci. USA, 83, 9694 (1986) and Sharon et al.,
`Science 234, 859 (1986)). The p55 or Tac chain and the p75
`chain each independently bind IL-2 with low or intermediate
`affinity, while the IL-2 receptor complex of both chains binds
`
`\
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`IL-2 with high affinity. The p75 chain of the human IL-2
`receptor will often be called herein simply the p75 protein.
`Much of the elucidation of the human IL-2 receptor's
`structure and function is due to the development of
`specifically reactive monoclonal antibodies.
`In particular,
`one mouse monoclonal antibody, known as anti-Tac (Uchiyama, et
`al., J. Immunol. 12.§., 1393 (1981)) has been used to show that
`IL-2 receptors can be detected on T-cells, but also on cells
`of the monocyte-macrophage family, Kupffer cells of the liver,
`Langerhans' cells of the skin and, of course, activated T(cid:173)
`cells.
`Importantly, resting T-cells, B-cells or circulating
`macrophages.typically do not display the IL-2 receptor
`(Herrmann, et al., J. Exp. Med. 162, 1111 (1985)). Another
`antibody, mik-,61, binds to the p75 chain (Tsudo et al.·, Proc.
`Nat. Acad. Sci. USA 86, 1982 (1989)).
`The anti-Tac monoclonal antibody has also been used
`to define lymphocyte functions that require IL-2 interaction,
`and has been shown to inhibit various T-cell functions,
`including the generation of cytotoxic and suppressor T
`lymphocytes in cell culture. Also, based on studies with
`anti-Tac and other ant~bodies, a variety of disorders are now
`associated with improper IL-2 receptor expression by T-cells,
`in particular adult T-cell leukemia.
`More recently, the IL-2 receptor has been shown to
`be an ideal target for novel therapeutic approaches to T-cell
`mediated diseases. It has been proposed that IL-2 receptor
`specific antibodies, such as the anti-Tac monoclonal antibody
`or mik-,61, can be used either alone or·as an·immunoconjugate
`(~ , with Ricin A, isotopes and the like) to effectively
`remove cells bearing the IL-2 receptor. These agents can, for
`example, theoretically eliminate IL-2 receptor-expressing
`leukemic cells, certain B-cells, or activated T-cells involved
`in a disease state, yet allow the retention of mature normal
`T-cells and their precursors to ensure the capability of
`·mounting a normal T-cell immune response as needed.
`In
`general, most other T-cell specific agents can destroy
`essentially all peripheral T-cells, which limits the agents'
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`therapeutic efficacy. Overall, the use of appropriate
`monoclonal antibodies specific for the IL-2 receptor may.have.
`therapeutic utility in autoimmune diseases, organ
`transplantation and any unwanted response by activated T-
`cells.
`Indeed, clinical trials have been.initiated using,
`Jh.9.:.., anti-Tac antibodies (Kirkman et al., Transplant. Proc.·
`ll, 1766 (1989) and Waldmann et al., Blood 72, 1805 (1988)).
`Unfortunately, the use of anti-Tac, mik-Pl ·and other'(cid:173)
`non-human monoclonal antibodies have certain drawbacks,
`particularly in repeated therapeutic regimens as explained ,
`below. Mouse monoclonal antibodies, for example, generally d9
`not fix human complement well, and lack other important
`immunoglobulin functional characteristics when used in humans.
`Perhaps more importantly, anti-Tac, mik-Pl and other non~huinan
`monoclonal antibodies contain substantial stretches of amino
`acid sequences that will be immunogenic when injected into a
`human patient.
`In accordance with the present invention, human-l~ke
`immunoglobulins specifically reactive with the p75 chain of\
`the human IL-2 receptor are provided. These immunoglobulins,
`which have binding affinities of at least 107 to 108 M-1
`, and
`prefe~~bly 10 9 M-1 to 10 10 M-1 or stronger, are capable of, ~ , \
`blocking the binding of IL-2 to human IL-2 receptors. The
`'.
`human-like immunoglobulins will have a human-like framework
`and can have complementarity dete~ining regions (CDR's) from
`an immunoglobulin, typically a mouse immunoglobulin,
`specifically reactive with an epitope on p75 protein. The
`immunoglobulins of the·present invention, which can be
`produced economically in large quantities, find use, for
`example, in the treatment of T-cell mediated disorders in
`human patients by a variety of techniques.
`In one aspect, the present invention is directed to
`recombinant DNA segments encoding the heavy and/or light c;::hain.
`CDR 1s from an immunoglobulin capable of binding to a desired
`epitope on the human IL-2 receptor, such as the mik-Pl
`monoclonal antibody. The DNA segments encoding these regions
`will typically be joined to DNA segments encoding appropriate
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`human-like framework regions. Exemplary DNA sequences, which
`on expression code for the polypeptide chains comprising the
`mik-Pl heavy and light chain CDRs, are included in Fig. 7.
`Due to codon degeneracy and non-critical amino-acid
`substitutions, other DNA sequences can be readily substituted
`for those sequences, as detailed above.
`The antibodies will typically find use individually
`in treating a T-cell mediated disease state. Generally, where
`the cell linked to a disease has been identified as IL-2
`receptor bearing, then the human-like antibodies capable of
`blocking the binding of IL-2 to the human IL-2 receptor are
`suitable{~, u.s.s.N. 085,707, entitled "Treating Human
`Malignancies and Disorders.") For example, typical disease
`states suitable for treatment include graft-versus-host
`disease and transplant rejection in patients undergoing an
`organ transplant, such as heart, lungs, kidneys, liver, etc.
`Other diseases include autoimmune diseases, such as Type I
`diabetes, multiple sclerosis, rheumatoid arthritis, systemic
`lupus erythematosus, and myasthenia gravis.
`The human-like antibodies of the present invention
`may also be used in combination with other antibodies,
`particularly human monoclonal antibodies reactive with other
`markers on cells responsible for the diseas·e.
`· For example,
`suitable T-cell markers can include those grouped into the so-
`called "Clusters of Differentiation," as named by the First
`International Leukocyte Differentiation Workshop, Leukocyte
`Typing, Bernard, et al., Eds., Springer-Verlag, N.Y. (1984).
`A preferred use is the simultaneous treatment of a patient
`with a human-like antibody binding to p55 and a human-like
`antibody binding to p75 of the IL-2 receptor, i.e., humanized
`anti-Tac plus humanized mik-p1.
`Human-like antibodies of the present invention can
`further find a wide variety of utilities in vitro. By way of
`example, the antibodies can be utilized for T-cell typing,
`for isolating specific IL-2 receptor bearing cells or
`fragments of the receptor, for vaccine preparation, or the
`like.
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`EXPERIMENTAL
`Cloning of heavy chain and light chain cDNA.
`cDNAs for the heavy chain and light chain variable domain
`genes were cloned using anchored polymerase chain reactions,
`(E.Y. Loh et al., science 243, 217 (1989)), using 3' primers ,
`that hybridized to the constant regions and contained HindIII
`sites, and 5 1 primers that hybridized to the dG tails and
`contained EcoRI sites (scheme shown in Fig. 6). The PCR
`amplified fragments were digested with EcoRI and HindIII and_
`cloned into the pUC19 vector for sequencing. For mik-Pl, two·
`gamma-2a specific and two kappa specific clones were
`sequenced. The two gamma-2a clones and two kappa clones are
`respectively identical in sequence. The cDNA variable domain
`sequences and the deduced amino acid sequences are shown in
`Fig. 7.
`
`Construction and expression of chimeric antibody.
`Two plasmid vectors were prepared for construction\
`and expression of the chimeric antibody genes. The plasmid
`pVgl-dhfr (Fig. SA) contains a·human cytomegalovirus IEl
`promoter and enhancer (M. Boshart et al., Cell 41, 521
`(1985)), the human genomic C~l segment including part of the.
`preceding intron, and a dihydrofolate reductase (dhfr) gene
`(Simonsen et al., Proc. Natl. Acad .. Sci. USA so, 2495 (1983))
`for selection. The plasmid pVk (Fig. SB) is similar to
`pVgl-dhfr but contains the human genomic c~ segment and the
`gpt gene. Derivatives of the mik-Pl heavy and light chain
`variable regions were prepared from the cDNAs by polymerase
`chain reaction. The 5' primers hybridized to the v regions
`starting at the ATG codons and contained XbaI sites; the 3'
`primers hybridized to the last 15 nucleotides of the J regions
`and contained splice donor signals and XbaI sites (.§ll, c.
`Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029 (1989)) .\
`The modified V regions were cloned into the XbaI sites of the
`respective plasmid vectors between the CMV promoter and the
`partial intrans of the constant regions.
`
`\
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`For expression of the chimeric antibody, the heavy
`chain and kappa chain plasmids were transfected into Sp2/0
`mouse myeloma cells by electroporation and cells selected for
`gpt expression. Clones secreting a m~ximal amount of.complete
`antibody were detected by ELISA. Purified chimeric mik-Pl
`antibody was shown to bind to YTJB cells, which express the
`p75 antigen, by flow cytometry (Fig. 9).
`
`Computer modeling of humanized antibodies.
`In order to retain high binding affinity in the
`humanized antibodies, the general procedures of Queen et al.
`were followed (C. Queen et al., Proc. Natl. Acad. Sci. USA 86,
`10029 (1989)). The more homologous a human antibody is to the
`original murine antibody, the less likely will combining the
`murine CDRs with.the human framework be to introduce
`distortions into the CDRs that could reduce affinity.
`Normally the heavy chain and light chain from the same human
`antibody are chosen to provide the framework seqeunces, so as
`to reduce the possibility of incompatibility in the assembling
`of the two chains. Based on sequence database (performed with
`the MicrorGenie Sequence Analysis Software (Beckman)), the
`antibody Lay was chosen to provide the framework sequences for
`humanization of mik-Pl.
`The computer program ENCAD (M. Levitt, J. Mol. Biol.
`168, 595 (1983)) was used to construct a model of the mik-Pl
`variable region. The model was used to determine the amino
`acids in the mik-Pl framework that were close enough to the
`CDRs to potentially interact with them (category 4 below). To
`design the humanized light and heavy chain mik-Pl variable
`regions, at each.position the amino acid was chosen to be the
`same as in the Lay antibody, unless th_at position fell in one
`or more of five categories:
`(1) The position fell within a CDR,
`(2) The Lay amino acid was unusual for human
`antibodies at that position, whereas the mik-Pl
`amino acid was typical for human antibodies at
`that position.
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`(3) The position was immediately adjacent to a CDR;,
`(4) The model described above suggested that the
`amino acid may be physically close to the
`antigen binding region (CDRs).
`For positions in these categories, the amino acid from the
`In addition, a position was.~
`(mouse) mik-Pl antibody was used.
`in the fifth category if
`(5) The Lay amino acid was highly unusual for human
`antibodies at that position, and the mik-P~
`amino acid was different but also unusual. ·.
`Then an amino acid typical for human antibodies
`at that position may be used.
`The amino acids in each category are shown in
`some amino acids may be in more than one category.
`Table 3.
`The final sequences of the humanized mik-Pl light and heavy
`chain variable domains are shown in Fig. 10, compared with the
`Lay sequences.
`
`Category
`1
`2
`
`3
`
`4
`5
`
`TABLE 3
`Light Chain
`24-33, 49-55, 88-96
`13
`
`70
`41
`
`Heavy Chain
`31-35, 50-65, 98-108'
`84, 89, 90
`30, 49
`29,· 30; 72, 73
`1
`
`For the construction of genes for the humanized
`antibodies, nucleotide sequences were selected that encode the
`protein sequences of the humanized heavy and light chains,
`including the same signal peptides as in the mouse mik-Pl.
`chains (Fig. 7), generally utilizing codons found in the mquse
`sequence. Several degenerate codons were changed to create·
`restriction sites or to remove undesirable ones~ The
`nucleotide sequences also included the same splice donor
`signals used in the chimeric genes and an XbaI site at each.
`end. Each gene was constructed from four overlapping
`synthetic oligonucleotides. For each variable domain gene,
`two pairs of overlapping oligonucleotides on alternating
`
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`strands were synthesized that encompassed the entire coding
`sequences as well as the signal peptide and the splice donor
`signal (Fig. 11). The oligonucleotides were synthesized on an
`Applied Biosystems 380B DNA synthesizer. Each oligo was about
`110-140 base long with about a 20 base overlap. Double
`stranded DNA fragments were synthesized with sequenase from
`each pair of oligonucleotides, digested with restriction
`enzymes, ligated to pBluescriptII KS(+) (Stratagene) vector
`and sequenced. Two fragments with the respectively correct
`half-sequences were then ligated into the Xb~I sites of the
`pVgl-dhfr or pVk expression vectors.
`In vitro mutagenesis was
`used to change an Ala amino acid originally encoded by
`oligonucleotide wps54 to the Glu (E) at position l of the
`humanized heavy chain (Fig. lOB) by changing the nucleotides
`CT to AG. Reactions were carried out under conditions well(cid:173)
`known in the art (Maniatis et al., ge .. _gjj;.)
`The heavy chain and light chain plasmids were
`transfected into Sp2/0 mouse myeloma cells by electroporation
`and cells were selected for gpt expression. Clones were
`screened by assaying human antibody production in the culture
`supernatant by ElISA, and antibody was purified from the best(cid:173)
`producing clones. Antibody was purified by passing tissue
`culture supernatant over a column of staphylococcal protein A(cid:173)
`Sepharose CL-4B (Pharmacia). The bound antibody was eluted
`with 0.2 M Glycine-HCl, pH3.0 and neutralized with 1 M Tris
`PHS.O. The buffer was exchanged into PBS by passing over a
`PD10 column (Pharmacia).
`
`Properties of humanized antibodies.
`The humanized mik-Pl antibody was characterized in
`comparison to the murine and chimeric antibodies. The
`humanized antibody bound to YTJB cells, which express p75
`chain at a high level, in a fluorocytometric analysis in a
`manner similar to the chimeric antibody (Fig. 9), showing that
`it recognizes the same p75 protein.
`The affinity of the humanized antibody was
`determined by competition with the radio-iodinated mouse
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`mik-Jfa antibody (Fig. 12). The binding affinities were
`calculated according to.the methods of Berzofsky (J.A.
`Berzofsky and I.J. Berkower, in Fundamental Immunology (ed~
`W.E. Paul), Raven Press (New York), 595 (1984)). The binding
`affinity of the humanized mik-Pl antibody was within about 2-
`fold of the affinity of the mouse mik-P1 antibody.
`The ability of humanized mik-Pl plus humanized anti-'
`Tac antibody (see, W090/07861 published July 26, 1990) to
`inhibit IL-2 stimulated proliferation of human lymphocytes was
`determined. Human mononuclear cells, collected from human
`blood by centrifugation on Ficoll-Paque (Pharmacia}, were.
`diluted to 2 x 106 cells/ml in RPM! medium+ 10% fetal calf
`serum (FCS). A 1/200 volume of phytohemagglutinin P (Difeo).
`was added and the cells were incubated for 4 days. The.cells
`were incubated an additionai 4 days in RPMI + 10% FCS + 10
`u/ml IL-2. 105 of these PHA activated blasts were then
`incubated with or without 2 µg each of humanized mik-Pl and· .
`humanized anti-Tac in 150 µl of RPMI + 10% FCS in wells of a.
`96-well plate for 1 hr, to which various dilutions of IL-2
`(Amgen) were then added in 50 µl medium. The cells were
`incubated 48 hr, 0.5 µCi methyl-3H-thymidine (Amersham, 82 Ci/
`mmol) was added, and the cells were incubated 24 hr. Cells
`were harvested with a cell harvester and radioactivity
`determined. The combination of the antibodies greatly
`inhibited proliferation of the cells in response to IL-2 (Fig.
`13), suggesting a combination of the antibodies will have
`strong immunosuppressive properties. Humanized mik-Pl plus
`humanized anti-Tac inhibited proliferation much more strongly
`than did either antibody alone.
`
`Example II
`Humanized Immunoglobulins to HSV Antigens
`Herpes Simplex Virus types I and II _(HSV-1 and Hsv~
`2) , are now estimated to be the second most frequent cause ot·.
`sexually transmitted diseases in the world. Although
`completely accurate data are not available, infection
`estimates range from about 20 to 40% of the U.S. population.·
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`A large number of diseases, from asymptomatic to
`life-threatening, are.associated with HSV infection. Of
`particular clinical interest, encephalitis from HSV-1
`infection and transmission of HSV-2 from a pregnant mother to
`her fetus are often fatal.
`Immunosuppressed patients are also
`subject to severe complications ·when infected with the virus.
`More than 50 HSV polypeptides have been identified
`in RSV-infected cells, including at least seven major cell
`surface glycoproteins (~, Whitley, R.~ Chapt. 66, and
`Roizman and Sears, Chapt. 65, Virology, Eds. Fields et al.,
`2nd ed., Raven Press, N.Y., N.Y. (1990)). The specific
`biologic functions of these glycoproteins are not .well
`defined, although gB and gD have been shown to be associated
`with cell fusion activity (W. Cai et al., J. Virol. 62, 2596
`(1988) and Fuller and Spear, Proc. Natl. Acad. Sci. USA 84,
`5454 (1987)). gB and gD express both type-specific and type(cid:173)
`common antigenic determinants. Oakes and Lausch demonstrated
`that monoclonal antibodies against gB and gE suppress
`replication of HSV-1 in trigeminal ganglia (Oakes and Lausch,
`J. Virol • .al., 656 (1984)). Dix et al. showed that anti-gc and
`gD antibodies protect mice against acute virus-induced
`neurological disease (D_ix et al., Infect. Immun. 34, 192
`(1981)). Whitley and colleagues produced a panel of murine
`monoclonal antibodies against HSV-1 and showed that several of
`the antibodies protected mice against encephalitis and death
`following ocular inoculation with the virus(~, Koga et al.,
`Virology 151, 385 (1986); Metcalf et al., cur. Eye Res.~, 173
`(1987) and Metcalf et al., Intervirology 29, 39 1988)). Clone
`Fd79 (anti~gB) prevented encephalitis even when immunization
`was delayed until 48 hours post-infection. Fd79 and Fdl38-80
`(anti-gD) significantly reduced the severity of epithelial
`keratitis and lowered the frequency of persistent viral
`infection in an outbred mouse model.
`In accordance with the present invention, humanized
`·immunoglobulins specifically reactive with HSV related
`epitopes either directly on the virus or on infected cells are
`provided. These immunoglobulins, which have binding
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`affinities to HSV specific antigens of at least about 107 M-1
`,
`and preferably 108 M""1 to 1010 M-1 or stronger, are capable of,·,
`~ , protecting cells from HSV transmission. The humanized
`·.
`immunoglobulins will have a human framework and will have one
`or more complementarity determining regions (CDR's) from ari
`immunoglobulin, typically a mouse immunoglobulin, specifica1·1y
`reactive with an HSV protein, such as gB and gD proteins. The
`immunoglobulins of the present invention, _which can be
`produced economically in large quantities, find use, for
`example, in the treatment of HSV mediated disorders in human
`patients by a variety of techniques.
`The HSVs are among the most intensively investigated
`of all viruses, and the HSV virion structure has been shown to
`contain about 33 proteins. Humanized immunoglobulins
`utilizing CDR's from monoclonal antibodies reactive with th~se
`proteins, particularly the eight surface glycoproteins (e.g.,
`gB, gC, gD, gE, gG, gH and gI), represent preferred
`embodiments of the present invention (~, Spear, P. G. , The·.
`Herpesviruses, vol. 3, pp. 315-356 (1984) (Roizman, B., ed), ·
`Plenum Press, N.Y., N.Y. and Spear, P.G., Immunochemistrv.of
`Viruses. The Basis for Serodiagnosis and Vaccines, pp. 425-
`446 (1985) (Neurath, A.R., eds.),.Amsterdam: Elsevier).
`In one aspect, the present invention. is directed to
`recombinant DNA segments encoding the heavy and/or light chairi
`COR's from an immunoglobulin capable of binding to a desired
`epitope of an HSV protein, such as monoclonal antibodies
`reactive with HSV gB and gD glycoproteins. The DNA segments·
`encoding these regions will typically be joined to DNA
`segments encoding appropriate humanized framework regions.·
`Exemplary DNA sequences code for the polypeptide chains
`comprising the heavy and light chain hypervariable regions
`.(with human framework regions) from monoclonal antibodies Fd79
`and Fdl38-80, shown in Fig. 14. Due to codon degeneracy and
`non-critical amino-acid substitutions, other DNA sequences can
`be readily substituted for those sequences, as detailed below.·.
`Any humanized immunoglobulins of the present
`invention may also be used in combination with other
`
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`antibodies, particularly humanized antibodies reactive with
`different HSV antigens. For example, suitable HSV antigens to
`which a cocktail of humanized immunoglobulins may react
`include gc, gE, gF, gG and gH (~, Rector, J. et al., Infect.
`Immun. 2§., 168 (1982) and·Fuller, A. et al., J. Virol. 63,
`3435 (1989)) •
`The antibodies can also be used as separately
`administered compositions given in conjunction with acyclovir
`or other antiviral agents. Typically, the agents may include
`idoxuridine or trifluorothymidine, but numerous additional
`agents (!h.!L., vidarabine) well-known to those skilled in the
`art for HSV treatment may also be utilized (see, Corey, L.,
`.QR..:. cit.). A preferred pharmaceutical composition of the
`present invention comprises the use of the subject
`immunoglobulins in immunotoxins to kill cells infected by HSV.
`These humanized antibodies can further find a wide
`variety of utilities .in vitro. By way of example, the
`antibodies can be utilized for detection of HSV antigens, for
`isolating specific HSV infected cells or fragments of the
`virus, for vaccine preparation, or the like.
`
`EXPERIMENTAL
`Cloning of heavy chain and light chain cDNA.
`cDNAs for the heavy chain and light chain variable
`domain genes were cloned using anchored polymerase chain
`regions (E.Y. Loh et al., Science 243, 217 (1989)), using 3'
`primers that hybridized to the constant regions and contained
`HindIII sites, and 5 1 primers that hybridized to the dG tails
`and contained EcoRI sites (scheme shown in Fig. 6). This
`method yields clones with authentic variable domain sequences,
`in contrast to other methods using mixed primers designed to
`anneal to the variable domain sequence (J.W. Larrick et al.,
`Bio/Technology 2, 934 (1989) and Y.L. Chiang et al., BioTech.
`1, 360 (1989)). The PCR amplified fragments were digested
`with EcoRI and HindIII and cloned into the pUC18 vector for
`sequencing. For Fd79, two gamma-1 specific and 5 kappa
`specific clones were sequenced. The two gamma-1 specific
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`clones are identical in sequence. This heavy chain cDNA
`fragment encodes a signal peptide of 19 amino acids, a V
`region in mouse heavy chain subgroup IIIB, a D segment, and a
`JBl segment with 4 alterations compared to the genomic JH1
`sequence. The deduced amino acid sequence is shown in Fig.
`14A.
`
`The five kappa specific clones belong to two groups.
`Two clones are identical and encode a kappa chain in which the
`conserved amino acid 23 cysteine has been substituted by a
`tyrosine, probably representing the non-productive allele •.
`The other three clones have an identical sequence encoding a
`signal peptide sequence of 20 amino acids, a V region in mouse
`kappa chain subgroup III, and a Jk2 segment with a single
`alteration compared_ to the genomic Jk2 sequence (Fig. 14B).
`The validity of the heavy chain and the kappa chain sequences
`was subsequently confirmed by the construction and expression
`of a chimeric antibody as discussed below.
`The heavy chain and the kappa chain of FdlJS-80 were.
`cloned similarly. Three clones each of the heavy chain and
`the kappa chain were sequenced~ All three heavy chain clon~s
`have an identical sequence encoding a signal peptide sequence.
`of 19.~ino acids, a V region in mouse heavy chain subgroup
`II, a D segment and the JH3 segment (Fig. 14C). The three·
`kappa clones are also identical in sequence. This light chain
`fragment encodes a signal peptide sequence of 20 amino acids;,
`a V region gene in mouse kappa chain subgroup V and the Jk5
`segment (Fig. 14D). Both chains shown no irregularities in.
`coding sequence; their validity was subsequently confirmed by
`construction and expression of a chimeric antibody.
`
`Construction and expression of chimeric antibodies.
`Two plasmid vectors were prepared for construction·.
`and expression of the chimeric antibody genes. The plasmid
`pVgl (Fig. 15A) contains a human cytomegalovirus IE1 promoter
`and enhancer (M. Boshart et al., Cell 41, 521 (1985)),·the
`human genomic c11 segment including part of the preceding
`intron, and the hygromycin gene (Blochlinger et al., Mol ..
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`Cell. Biol.~, 2929 (1984)) for selection. The plasmid pVk
`(Fig. lSB) is similar to pVgl but contains the human genomic c~
`segment and the gpt gene. Derivatives of the Fd79 and Fd138-
`80 heavy and light chain variable regions were prepared from
`the cDNAs by polymerase chain reaction. The 5 1 primers
`hybridized to the V regions starting at the ATG codons and
`contained XbaI sites; the 3' primers hybridized to the last 15
`nucleotides of the J regions and contained splice donor
`signals and XbaI sites(~, c. Queen et al., Proc. Natl.
`Acad. Sci. USA 86, 10029 (1989)). The modified V regions were
`cloned into the XbaI sites of the respective plasmid vectors
`between the CMV promoter and the partial introns of the
`constant regions.
`For expression of the chimeric antibodies, the heavy
`chain and kappa chain plasmids were transfected into Sp2/0
`mouse myeloma cells by electroporation and cells selected for
`gpt expression. Clones secreting
`a maximal amount of complete
`antibody were detected by ELISA.
`Purified chimeric Fd79 and
`Fdl38-BO antibodies were shown to
`bind to HSV-1 infected vero
`cells by flow cytometry. Viral neutralization assays also
`indicated that the chimeric antibodies retain the
`neutralization activities of the murine antibodies (data not
`shown, but see below for similar results with humanized
`antibodies).
`
`Computer modeling of humanized antibodies.
`In order to retain high binding affinity in the
`humanized antibodies, the general procedures of Queen et al.
`were followed (C. Queen et al., Proc. Natl. Acad. Sci. USA 86,
`10029 (1989)). The more homologous a human antibody is to the
`original murine antibody, the less likely will combining the
`murine CDRs with the human framework be to introduce
`distortions into the CDRs that could reduce affinity.
`Normally the heavy chain and light chain from the same human
`antibody are chosen to provide the framework sequences, so as
`to reduce the possibility of incompatibility in the assembling
`of the two chains. Based on sequence homology search against
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`the NBRF protein sequence database (performed with the
`MicroGenie Sequence Analysis Software (Beckman)), the antibody.
`Pom was chosen to provide the framework sequences for
`humanization of Fd79.
`The computer program ENCAD (Levitt, J. Mol. Biol.
`168, 595 (1983)) was used to construct a model of the Fd79
`variable region.
`Inspection of the refined model of murine
`Fd79 revealed two amino acid residues in the framework that
`are close enough to have significant contacts with the CDR
`residues (Table 4). Lys in light chainNBRF positi